Even when it is possible to prevent hyperacute rejection (HAR) of vascularized xenografts across discordant species, such as pig to human cellular responses may lead to delayed xenograft rejection (DXR). Evidence is mounting that human natural killer (huNK) cells may play a major role in DXR through activation of endothelial cells (EC). Furthermore, huNK cells can lyse pig EC through antibody- dependent dell-mediated cytotoxicity (ADCC), when triggered by xenoreactive natural antibodies (XNA). Recognition of self major histocompatibility complex class I (MHC-1) molecules by killer inhibitory receptors (KIRs, CD94) appears to be the major mechanism for down regulating NK cytotoxic activity. The factors and receptors that promote huNK cell activation or inhibition by pig EC are not well understood. The immediate goal of this proposal, therefore, is to develop methods of inhibiting anti-pig huNK cytotoxicity, by engineering human MHC-1 (HLA) molecules to block anti-pig huNK cell activation. The long range goal is to make it possible to transplant pig tissues into humans without eliciting xenoreactive cellular cytotoxicity. The specific aims are: 1) To test the ability of individual HLA molecules, HLA homologues, or cell-surface anti-KIR antibodies, to provide partial protection of transfected pig EC against killing by polyclonal huNK cells, and complete protection against phenotypically different huNK clones. These experiments will characterize anti-pig huNK cell KIRs at a clonal level, and suggest ligand combinations that might inhibit the polyclonal response. 2) To test the ability of select combinations of inhibitory ligands characterized under Aim 1, to provide complete protection against killing by polyclonal huNK cells. 3) To test the hypothesis that inhibitory signals transduced through KIR/CD94 recognition of transfected ligands will block XNA- mediated ADCC by anti-pig hunk cells. 4) To test the ability of genetically engineered "next generation" isoforms of inhibitory HLA molecules to prevent lysis by human cytotoxic T lymphocytes (CTLs) reacting to transfected allogeneic HLA, while retaining their ability to block killing by anti-pig huNK cells. To diminish alloreactivity, HLA molecules will be engineered so they are soluble or glycosyl phosphatidylinositol (GPI)-anchored or unable to interact with the CD8 and/or the peptide transporter TAP. These in vitro experiments will lay the groundwork for a strategy to inhibit pig to human xenograft rejection in vivo.